Incubating shakers are used to serve reaction flasks and their content in providing desired temperature, stirring, mixing and resulting gas-to-liquid mass transfer through either rotary or reciprocating shaking motion for nearly half a century. Both have throw motions normally in range of 12.5 to 50 mm, i.e., 0.5-2 in, the maximum linear distance moved by any point in and on the flask. Reciprocation is usually about 100 cycles/min and in rotary shaking the range is about 150 to 300 rev/min (Pirt, 1975). The mechanical drive and shaft mechanism causes the shaker table and flask vessels clamped on to the table to gyrate, orbit or move thereby causing the shaker table and flask vessels to shake. When used in microbial fermentation industry, for example, the objectives are usually two folds. One is to provide the key link in translating laboratory culture data to commercial scale operation, i.e., scale up; and the other in scaling down environmental conditions achievable in commercial scale equipment to this laboratory and frequently bench size equipment. Both are to insure that improvement studies are carried out under conditions that can be duplicated in either direction. (Aiba, Humphrey and Millis, 1973)
Shaken flasks' main utility is in the comprehensive change of reaction conditions from one flask to another and/or from one incubating shaker to another, e.g., substrate concentration, temperature, reacting species/formulation, mixing power, etc. The simplicity of its preparation and operation, and the economy in time, material, flask acquisition, and hence number advantage for repeated runs have made shaken flask a work horse and its unshaken generic role in science and engineering labs. (Betts and Baganz, 2006; Kumar, Wittmann and Heinzle, 2004)
One well known drawback of shaken flask (often conical Erlenmeyer flasks) is its limited atmospheric gas exchange or ventilation when reaction must be shielded from the ambient to avoid contamination, notably the use of cotton or sponge plug or gauze/cotton/gauze “sandwiched” layers for closuring from air-born contaminants when culturing things like microbes. Here, atmospheric exchange between flask headspace and ambient incubator gas is limited to natural convection or diffusion resulting from concentration gradient across the porous gas-diffusible closuring. Blocked by the porous or spongy closure, gaseous reaction product tends to get concentrated in flask headspace, while feeder or substrate gas stayed out. In aerobic microbial culture this may result in reduced cell growth and the consequent reaction rate due to oxygen starvation and/or CO2 inhibition. The same is true with the openings, closuring or caps of other static and/or shake culture vessels such as test tubes, tissue culture T-flasks, micro-titer plate, etc. (Betts and Baganz, 2006; Kumar, Wittmann and Heinzle, 2004).
This becomes less a problem when larger and more sophisticated reaction vessels like standard stirred tank reactor (STR) are used. They solve this gas exchange or ventilation problem by forced ventilation such as use of direct sparging, membrane permeation, reaction chamber pressurization, gas pumping, etc. to supply the substrate gas, and in turn purge the waste or product gas out of the vessel. During direct gas sparging in a standard STR, substrate-gas bubbles are injected toward and chopped and dispersed by the high speed impeller blades. The resulting fine gas bubbles not only increase the volume of gas holdup in the liquid phase, but also provide expanded gas-liquid interfacial area for enhanced gas transfer into solution. Forced ventilation or gas sparging using pressurized line gas supplies fresh feeder gas and purges inhibitory waste product gas like oxygen and CO2, respectively, inside a microbial culture vessel, to facilitate higher rate of reaction such as faster aerobic cell growth.
However, forced ventilation, using pressurized line gas, on smaller size reaction vessel like shaken flask is not easily workable without compromising its aforementioned advantage of simplicity and economy in time, material, acquisition, and number. Hence, gas supply, exchange or ventilation in enclosed reaction vessels like flasks, bottles, beakers, tubes, micro-titer plate wells, etc. (they are all termed “reaction vessel” below) in number in floor or bench-top scale shaker or mixer is still without a solution which can combine the best of shaken flask and STR. Available solutions in tissue cell and/or microbial culture see modifications of vessel closuring cap for sterile venting and breathing of tissue culture flask (TPP/MIDSCI Tissue Culture Products from BD Falcon; Eudailey and Lyman, 2007), of tissue culture flask compartmentalization for better maintenance of high cell density (Wilson and Wolf, 1997), of microbial fermentation flasks' shape and locations of their membraned “windows” for gas exchange capacity (Kato and Tanaka, 1998), of improved microbial flask baffling and closuring for enhanced aeration (Tunac, 1987), of system for sparged aeration of six 500 ml microbial flasks on shaker (Donovan, Robinson and Glick, 1995), of gas delivering fittings on tissue culture spinner flask for forced gas supply and aeration (ProCulture Spinner Flasks from Corning), and of single-use hybrid-mode bag bioreactor using sparge tube and stirring propellers for STR-like mixing and aeration (CellMaker PLUS from Cellexus Biosystems). These improvements all but still rely basically on either natural convection thru the vessel closurings like the shake flask or forced gas flow by sparging like the STR. The patent literature by Tunac (1987) and journal article by Kato and Tanaka (1998) in particular addressed the same problem as this invention, but only went as far as with shaker-motion-enhanced local gas “disturbance” in and around the modified venting cap or the membraned “windows” of the flask, and without the sustained and controlled fresh gas supply and purging within and the quantitative proof emphasized in present invention.
Extensive search of patents, scientific journals and Internet content databases reveal no prior design, use or application meeting the functional criteria of sustained convective flow of fresh gas intake and spent gas vent with aid of indigenous liquid mixing and without use of line gas in present invention. Known laboratory shake flask, incubator shaker and bioreactor suppliers also do not carry product meeting these criteria. Recent reviews of relevant prior art were authored by Betts and Baganz (2006) on miniature bioreactors and by Kumar, Wittmann and Heinzle (2004) on minibioreactors.